RESUMEN
RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-α) or alpha interferon (IFN-α) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short double-stranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-α production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-α, independent of 5'-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/inmunología , Gripe Humana/virología , Inosina/genética , Edición de ARN , ARN Viral/genética , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 8/inmunología , Adenosina/genética , Adenosina/inmunología , Adenosina Desaminasa/genética , Adenosina Desaminasa/inmunología , Animales , Secuencia de Bases , Femenino , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/genética , Inosina/inmunología , Interferón-alfa/genética , Interferón-alfa/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/inmunología , Receptor Toll-Like 7/genética , Receptor Toll-Like 8/genéticaRESUMEN
STUDY DESIGN: In vivo study using a moderate spinal cord contusion injury (SCI) model in adult rat. OBJECTIVE: To assess the immunomodulatory effects of the purine nucleoside inosine on macrophage/microglia activation at and near the lesion site and in white matter areas remote from the injury epicenter. SETTING: Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY, USA. METHODS: Animals (N=56) were injured using a moderate SCI at T9-T10 spinal level and were divided into three groups, depending on treatment paradigm. Rats received either intraperitoneal or subcutaneous injections of inosine (N=28) or vehicle (N=28). Spinal cord tissue was processed for ED-1 immunoreactivity and the volume fraction of ED-1(+) profiles was calculated using the Cavalieri method and unbiased stereology. RESULTS: The volume fraction of ED-1(+) profiles within gray and lateral white matter regions at and around the lesion site was significantly reduced only following a twice daily-6 week treatment course, compared with vehicle controls, and white matter areas remote from the lesion were unaffected by all inosine treatment paradigms. CONCLUSIONS: Continued subcutaneous delivery of inosine, beginning 15-min post-SCI and persisting throughout the survival period of 6 weeks exerted immunomodulatory effects at and around the lesion site.
Asunto(s)
Factores Inmunológicos/farmacología , Inosina/farmacología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/inmunología , Médula Espinal/efectos de los fármacos , Médula Espinal/inmunología , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/inmunología , Modelos Animales de Enfermedad , Femenino , Gliosis/tratamiento farmacológico , Gliosis/inmunología , Gliosis/prevención & control , Factores Inmunológicos/inmunología , Factores Inmunológicos/uso terapéutico , Inyecciones Intraperitoneales , Inyecciones Subcutáneas , Inosina/inmunología , Inosina/uso terapéutico , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Microglía/efectos de los fármacos , Microglía/inmunología , Nucleósidos de Purina/inmunología , Nucleósidos de Purina/farmacología , Nucleósidos de Purina/uso terapéutico , Ratas , Ratas Long-Evans , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Resultado del TratamientoRESUMEN
Synthetic phosphorothioate oligodeoxynucleotides (ODN) bearing unmethylated CpG motifs can mimic the immune-stimulatory effects of bacterial DNA and are recognized by Toll-like receptor 9 (TLR9). Past studies have demonstrated that nucleotide modifications at positions at or near the CpG dinucleotides can severely affect immune modulation. However, the effect of nucleotide modifications to stimulate human leukocytes and the mechanism by which chemically modified CpG ODN induce this stimulation are not well understood. We investigated the effects of CpG deoxyguanosine substitutions on the signaling mediated by human TLR9 transfected into nonresponsive cells. ODN incorporating most of these substitutions stimulated detectable TLR9-dependent signaling, but this was markedly weaker than that induced by an unmodified CpG ODN. One of the most active ODN tested contained deoxyinosine for deoxyguanosine substitutions (CpI ODN), but its relative activity to induce cytokine secretion on mouse cells was much weaker than on human cells. The activity was dependent on TLR9, as splenocytes from mice genetically deficient in TLR9 did not respond to CpI ODN stimulation. It is surprising that CpI ODN were nearly as strong as CpG ODN for induction of human B cell stimulation but were inferior to CpG ODN in their ability to induce T helper cell type 1 effects. These data indicate that certain deoxyguanosine substitutions in CpG dinucleotides are tolerated to stimulate a TLR9-mediated immune response, but this response is insufficient to induce optimal interferon-alpha-mediated effects, which depend on the presence of an unmodified CpG dinucleotide. These studies provide a structure-activity relationship for TLR9 agonist compounds with diverse immune effects.
Asunto(s)
Linfocitos B/efectos de los fármacos , Islas de CpG/inmunología , Inosina/análogos & derivados , Activación de Linfocitos/efectos de los fármacos , Glicoproteínas de Membrana/efectos de los fármacos , Oligodesoxirribonucleótidos/farmacología , Receptores de Superficie Celular/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Secuencias de Aminoácidos/inmunología , Animales , Linfocitos B/inmunología , Línea Celular , Femenino , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/inmunología , Compuestos Heterocíclicos/farmacología , Humanos , Inosina/química , Inosina/inmunología , Inosina/farmacología , Interferón-alfa/inmunología , Activación de Linfocitos/inmunología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Estructura Molecular , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/inmunología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T/inmunología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Receptor Toll-Like 9 , Receptores Toll-LikeRESUMEN
A highly sensitive enzyme-linked immunoassay (ELISA) was developed to detect and quantify the tumor marker, 1-methylinosine (m1I), in human urine. The rabbit antisera was highly specific for m1I with negligible or no inhibition by other nucleosides excreted into urine. Using the competitive ELISA, nanogram amounts of m1I were easily measured directly in urine. The assay agreed with our previous hplc analysis of m1I in urine for identifying those individuals with chronic myelogenous leukemia. Thus, this assay should greatly facilitate the quantitation of m1I as a tumor marker.